Active transport of Na+ and K= across the cell membrane is catalyzed by the Na,K-ATPase, an enzyme which affects the physiology of nervous tissue in several interrelated ways. Previous work (Sweadner 1979) described the identification and separation of two different Na,K-ATPases in the mammalian nervous system. The two enzymes have structural differences and can be isolated from different kinds of cells: one is found in astrocytes, sumpathetic neurons, cardiac and skeletal muscle, while the other is found axolemma from myelinated nerve. The proposal describes biochemical and immunological approaches to discover their different functional roles and distribution. The two Na,K-ATPases will be purified, and their subunits identified by gel electrophoresis and cross-linking. Functional differences of two kinds will be examined: First, the ratio of Na+ exchanged for K+ will be measured with enzyme reconstituted into lipid vesicles. The ratio determines the electrogenic effect of the pump on cell membrane potential. Second, the regulation of ion ttansport by each enzyme will be compared. Preliminary results suggest a mechanism for ion transport regulation involving the uncoupling of transport from ATPase activity. The hypothesis is that uncoupling of this Na,K-ATPase results from a modification controlled by biological factors. The biochemical basis of the proposed modification will be determined, and then the coupling of the Na,K-ATPase will be measured in lipid vesicles. Finally, the stimulation of ion transport by nerve growth factor will be used to test the hypothesis in intact cells. The cellular distsribution of the two Na,K-ATPases will be determined by immunofluorescence. Antibodies specific for the two Na,K-ATPases will be purified from sera raised in rabbits. Localization studies will be done on the retina, a portion of the nervous system that lends itself to future studies of the functional differences between the two Na,K-ATPases in situ. It is hoped that the outcome of the research will be the assignment of different ion transport control mechansms to different kinds of cells, information which will b integrated into the larger effort of understanding nervous system function.